Transgenic algae for delivering antigens to an animal

ABSTRACT

Delivery systems and methods are provided for delivering a biologically active protein to a host animal. The systems and methods provided include obtaining an algal cell transformed by an expression vector, the expression vector comprising a nucleotide sequence coding for the biologically active protein, operably linked to a promoter. In one illustrated embodiment, the biologically active protein is an antigenic epitope and upon administration to the animal the algal cell induces an immune response in the host animal.

FIELD OF THE INVENTION

This invention relates to a system for delivering antigens to an animal.

BACKGROUND OF THE INVENTION

Proteins, dipeptide and polypeptide (hereinafter collectively referredto as proteins) are responsible for most of the activities of a cell,such as catalysis, communication, defense, movement, and transport.

Proteins can be delivered to animals for the purpose of activating orsupplementing a biological activity. Examples include antigens thatactivate an immune response and hormones that regulate growth anddevelopment.

The bases of a protein's biological activity is its sequence and/orconformation. Hence, the biologically active portion (such as anepitome) should remain essentially intact until it reaches its targetdestination. Factors that could limit the biological activity of aprotein include chemical and enzymatic denaturation, as well asstructural barriers that preclude entry into the animal or access to thetarget destination.

The patent describes a method for producing, and then deliveringbiologically active proteins to an animal using transgenic algae. Thedelivery of an antigen to an animal and activation of an immune responseis offered as a specific example.

Infectious Disease in Humans

Infectious disease is an age-old problem. Early in human history,infectious diseases such as smallpox, bubonic plague, influenza, measlesand many others caused epidemics and countless deaths. More recently,epidemics of Legionnaires' disease, human immunodeficiency virus, Ebolavirus, Lyme disease, and others have been significant threats to humanhealth.

Solutions to Infectious Disease

Early in human history, quarantine of infected individuals and improvedsanitation measures were used to decrease the spread of infectiousdisease. Later, chemotherapeutic agents (drugs) were invented and, earlyon, included chemicals like sulfur and mercury. Modern chemotherapeuticsinclude antibiotics, antiviral drugs, and antiparasitic drugs. Althoughessential, there are properties of such drugs that are not ideal. Forexample, drugs do not prevent disease. Rather, drugs are administeredafter a disease is diagnosed. Another problem is that infectious agentscan develop resistance to the drugs such that the drugs are no longereffective against the infectious agents. Finally, drugs can causeserious side affects in the individual to which they are administered.

An alternative to chemotherapeutic agents is administration ofimmunogenic agents, wherein the immunogens that comprise a compositionoriginating from the infectious agent whose infection one is trying toprevent. After administration, such immunogenic compositions preferablystimulate the immune system such that subsequent infection by theinfectious agent is prevented, or disease symptoms caused by theinfectious agent are decreased. Such immunogenic compositions areadvantageous in that they are administered before the individualcontracts the infectious agent. The stimulation of immunity in theindividual caused by administration of the immunogenic composition,therefore, is designed to prevent infection and disease. Suchimmunogenic compositions normally produce few side effects in theindividual to which the composition is administered. Finally, infectiousagents do not normally develop resistance to immunity that develops as aresult of administration of the antigen composition.

Infectious Disease in Non-Human Animals

Infectious diseases in non-human animals also cause significantmorbidity and mortality. Such disease is important, not only becausenon-human animals can sometimes transmit the infectious agents tohumans, but also because non-human animals and the products thereof areimportant human food sources and their loss is economically burdensome.

Infectious Diseases in Aquaculture

An example of an area where infectious non-human animal diseasescontinue to affect an important human food source is aquaculture.Aquaculture is the fanning of aquatic organisms, including fish andother seafood, for human consumption. Currently in the U.S., thedomestic fishing industry meets only a small part of the total demandfor fish. In 1997, for example, the federal trade deficit for importedfish was nearly $9 billion, the third largest component of the U.S.foreign trade deficit.

In an attempt to meet the demand for fish, the aquaculture industry hasresponded and, in 1997, produced over $55 billion in farmed fish(statistic from the Food and Agriculture Organization of the UnitedNations). Furthermore, the aquaculture industry has grown, historicallybetween 10-20% per year for the last ten years. Therefore, it is clearthat aquaculture is a rapidly emerging supplement and replacement forthe traditional fishing industry and has tremendous growth potential.

One of the major constraints for aquaculture, however, is disease. Underthe high density conditions under which fish and other aquatic organismsare farmed, the incidence of infectious disease can be high and, whendisease does occur, it can spread rapidly through entire populationswith high mortality. On average, 10-30% of farmed fish production, andup to 80% of shrimp production, is lost due to disease (Austin B., etal., 1987 Bacterial Fish Pathogens: Disease in Farmed and Wild Fish, 364pp Publishers: (Ellis Horwood Ltd., Chichester, UK)).

Solutions to Infectious Diseases in Aquaculture

Again using aquaculture as a specific example, fish diseases with abacterial etiology can be effectively treated with chemotherapeuticagents from the class called antibiotics. However, as much as 80% of theantibiotic may pass through the fish (Pothuluri, et al., 1998, Res. Dev.Microbiol. 2:351-372), and development of bacteria resistant to theantibiotic may also arise. Such antibiotic-resistant bacterial pathogenscan spread, creating entire fish populations harboring pathogenicbacteria that are resistant to the antibiotic. Clearly, it would beadvantageous to prevent infection of the fish by the bacteriaaltogether. Another consideration is that viral and parasitic diseasescannot be treated with antibiotics.

Another strategy for preventing infection and reducing fish losses dueto disease is prophylactic administration of an antigen composition,wherein the antigens are derived from an infectious agent, to stimulatethe immune system of fish, other aquatic organisms, or other organismsgenerally (Gudding, et al., 1999, Vet. Immun. Immunopath. 72: 203-212).

Methods of Introducing Antigens into Animals

One problem with antigen compositions, especially in fish, is that manymethods for administering them may not be technically or economicallypractical. For example, direct injection of the antigen composition intofish is labor intensive and is often expensive relative to the futuremarket value of the fish. Furthermore, injection needles cancross-infect fish with contaminating infectious agents, and accidentalinjection of humans can cause severe infections and anaphylacticreactions. In addition, noninjurious injection of small fish may bedifficult.

An alternative route of administration is an oral method wherein theantigen composition is incorporated into the fish food, for example.Another improved method of administering antigens to fish is immersionof the fish for a preset period of time in a suspension of the antigen.However, it can be costly to produce, purify, and package the antigensfor such use. Prior art methods of producing antigens have involved thedifficulty of growing fish viruses in culture systems to produce enoughvirus to obtain a sufficient quantity of antigen. In addition, antigencompositions to be administered orally are often encapsulated inexpensive polysaccharide-coated beads. Finally, oral administration ofantigen compositions have previously shown low and inconsistent levelsof stimulation of the fish immune system, thereby minimizing protectionagainst subsequent infection by the infectious agent.

SUMMARY OF THE INVENTION

In accordance with the present invention, a delivery system is providedfor delivering peptides to a host animal. The peptides may be growthhormones, antigens derived from a pathogen, other antimicrobialpeptides, etc. The delivery system is a transgenic algae that comprisesa transgene which comprises a) a polynucleotide encoding at least onepeptide, for example an antigenic determinant for the pathogen, and b) apromoter for driving expression of the polynucleotide in the algae. In apreferred embodiment, the transgene further comprises c) a terminatorthat terminates transcription, and d) all other genetic elementsrequired for transcription. In another preferred embodiment, thetransgenic algae further expresses the peptide. If the peptide is anantigenic determinant it is preferably located on the cell surface orwithin the cytoplasm or an organelle of the transgenic algae. Thetransgenic algae of the present invention is useful for inducing animmune response in the host animal to the pathogen. The transgenic algaeof the present invention is also for treating, ameliorating, orpreventing a disease caused by the pathogen.

The present invention also provides methods for delivering the peptide,for example, an antigenic determinant derived from a pathogen, to a hostanimal. In one aspect, the method comprises orally administering atransgenic algae which comprises (a) a polynucleotide that encodes atleast one antigenic determinant of a pathogen for the host animal and(b) a promoter that drives expression of the polynucleotide in thenucleus or an organelle of the algae. Such method is especially usefulfor delivering the antigenic determinant to a mammal or an aquaticanimal. In another aspect, the method comprises immersing the hostanimal into a suspension comprising water and a transgenic algae whichcomprises (a) a polynucleotide that encodes at least one antigenicdeterminant of a pathogen for the host animal, and (b) a promoter thatdrives expression of the polynucleotide in the nucleus or an organelleof the algae. Such method is especially useful for delivering theantigenic determinant to an aquatic animal.

Thus, in one aspect of the invention, a delivery system is provided fordelivering a biologically active protein to a host animal comprising analgal cell transformed by an expression vector, the expression vectorcomprising a nucleotide sequence coding for the biologically activeprotein, operably linked to a promoter.

In another aspect of the invention, a delivery system is provided fordelivering antigens to a host animal comprising an algal celltransformed by an expression vector, the expression vector comprising anucleotide sequence coding for an antigenic determinant.

In still another aspect of the invention, a method is provided forinducing an immune response in an animal, comprising the steps ofobtaining a transgenic alga expressing an antigenic peptide,administering the transgenic alga to the animal.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing figures wherein:

FIG. 1 shows the pSSCR7 plasmid used for constructing nucleartransfection vectors for use in the present invention;

FIG. 2 shows the pCPPTG plasmid used for constructing chloroplasttransfection vectors for use in the present invention;

FIG. 3 is a diagram showing the expression between the 3′ end of a lowCO₂-induced plasma-membrane protein gene and a P57 antigen, and showingthat the P57 antigen is located on the periplasm side of the cellmembrane;

FIG. 4 is a western blot of trout sera probed against algal cells usedfor oral immunization. Lane 1: E-22; Lane 2: CP57; Lane 3: CC-2137(WT);Lane 4: E-22; Lane 5: CP57; Lane 6: CC-2137(WT); Lane 7: E-22; Lane 8:CP57; Lane 9: CC-2137(WT); Lane 10: E-22; Lane 11: CP-57; Lane 12:CC-2137(WT); and

FIG. 5 is a western blot of Trout Mucus Probed Against Algal Cells usedfor Immersion Immunization. Lane 1: E-22; Lane 2: CP57; Lane 3:CC-2137(WT); Lane 4: E-22; Lane 5: CP57; Lane 6: CC-2137(WT); Lane 7:E-22; Lane 8: CP57; Lane 9: CC-2137(WT); Lane 10: E-22; Lane 11: CP-57;Lane 12: CC-2137(WT);

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention provides a delivery system forintroducing one or more peptides into a host animal. The delivery systeminvolves the use of is a transgenic algae comprising a transgene whichcomprises a polynucleotide encoding one or more peptides and a promoterwhich drives expression of the peptide encoding sequence in the algae.

Antigenic Determinant

The term “antigenic determinant” as used herein refers to a protein orpolypeptide which is capable of eliciting an immune response or defenseresponse in the host animal. The antigenic determinant is at leastpartially derived from a pathogenic microorganism. Herein, the term“microorganism” means a bacterium, virus, fungus, or parasite (i.e.,protozoan or helminth, for example). Preferably, the immune response ordefense response elicited by the antigenic determinant is protective inthe host animal in the sense that a subsequent infection of the hostanimal by the pathogenic organism from which the antigenic determinantis derived would be prevented, would not cause disease or, if diseasewere caused, the disease or symptoms associated with the disease wouldbe ameliorated. Preferably, the antigenic determinant itself does notcause disease or any other adverse symptoms in the host animal.

The antigenic determinant is either a holoprotein or a portion of aprotein that stimulates the immune system of an animal that is a hostfor the pathogenic organism. Typically, the antigenic determinants areeither secreted by the pathogen or is found on the cell membrane or cellwall thereof, but could potentially be any component of the pathogen.The antigenic determinant may be part of fusion protein. For example, itmay be advantageous to fuse the antigenic determinant with a proteinthat is expressed on the surface of the algal cell.

The transgenic algae of the present invention comprise at least oneantigenic determinant of a pathogenic microorganism. In certainembodiments the present transgenic algae comprise a plurality ofantigenic determinants from a single microorganism. In otherembodiments, the present transgenic algae comprise one or more antigenicdeterminants from a plurality of pathogenic microorganisms. Whenexpressed, the antigenic determinants may be located in the cytoplasm,in an organelle, particularly the mitochondria or chloroplast, on thecell surface, exported from the cell or in a combination of locations inthe algae.

Host Animals

As used herein the term “host animal” refers to all animals capable ofmounting an immune or defense response when infected with a pathogenicmicroorganism. Accordingly, the term host animal, as used herein,encompasses mammals, including humans, companion animals such as catsand dogs, non-companion animals such as cattle and sheep, birds, aquaticvertebrates, and aquatic invertebrates.

Because algae are a food substance for numerous aquatic animals, thetransgenic algae of the present invention are especially useful fordelivering an antigenic determinant to aquatic vertebrates andinvertebrates. Such aquatic vertebrates include, but are not limited to,all vertebrate fish, which may be bony or cartilaginous fish. Suchfin-fish include but are not limited to salmonids, carp, catfish,yellowtail, seabream, and seabass.

Immune systems in fish are essentially the same as the immune systems ofmammals. The immune system is organized into discrete compartments toprovide the milieu for the development and maintenance of effectiveimmunity. Those two overlapping compartments: the lymphoid andreticuloendothelial systems (RES) house the principal immunologic cells,the leukocytes. Leukocytes derived from pluripotent stem cells in thebone marrow during postnatal life include neutrophils, eosinophils,basophils, monocytes and macrophages, natural killer (NK) cells, and Tand B lymphocytes. Hematopoietic and lymphoid precursor cells arederived from pluripotent stem cells. Cells that are specificallycommitted to each type of leukocyte (colony-forming units) areconsequently produced with the assistance of special stimulating factors(e.g., cytokines).

Leukocytes, the main cells in the immune system, provide either innateor specific adaptive immunity. These cells are derived from myeloid orlymphoid lineage. Myeloid cells include highly phagocytic, motileneutrophils, monocytes, and macrophages that provide a first line ofdefense against most pathogens. The other myeloid cells, includingeosinophils, basophils, and their tissue counterparts, mast cells, areinvolved in defense against parasites and in the genesis of allergicreactions. In contrast, lymphocytes regulate the action of otherleukocytes and generate specific immune responses that prevent chronicor recurrent infections.

Lymphoid Cells provide efficient, specific and long-lasting immunityagainst microbes and are responsible for acquired immunity. Lymphocytesdifferentiate into three separate lines: thymic-dependent cells or Tlymphocytes that operate in cellular and humoral immunity, B lymphocytesthat differentiate into plasma cells to secrete antibodies, and naturalkiller (NK) cells. T and B lymphocytes are the only lymphoid cells thatproduce and express specific receptors for antigens. T Lymphocytes areinvolved in the regulation of the immune response and in cell mediatedimmunity and help B cells to produce antibody (humoral immunity). MatureT cells express antigen-specific T cell receptors (TcR) that areclonally segregated (i.e., one cell lineage-one receptor specificity).Every mature T cell also expresses the CD3 molecule, which is associatedwith the TcR. In addition mature T cells display one of two accessorymolecules, CD4 or CD8. The TcR/CD3 complex recognizes antigensassociated with the major histocompatibility complex (MHC) molecules ontarget cells (e.g. virus-infected cell). The TcR is a transmembraneheterodimer composed of two polypeptide chains (usually, alpha and betachains). Each chain consists of a constant (C) and a variable (V)region, and is formed by a gene-sorting mechanism similar to that foundin antibody formation. The repertoire is generated by combinatorialjoining of variable (V), joining (J), and diversity (D) genes, and by Nregion (nucleotides inserted by the enzyme deoxynucleotidyl-transferase)diversification. Unlike immunoglobulin genes, genes encoding TcR do notundergo somatic mutation. Thus there is no change in the affinity of theTcR during activation, differentiation, and expansion.

The activation of B cells into antibody producing/secreting cells(plasma cells) is antigen-dependent. Once specific antigen binds tosurface Ig molecule, the B cells differentiate into plasma cells thatproduce and secrete antibodies of the same antigen-binding specificity.If B cells also interact with Th cells, they proliferate and switch theisotype (class) of immunoglobulin that is produced, while retaining thesame antigen-binding specificity. This occurs as a result ofrecombination of the same Ig VDJ genes (the variable region of the Ig)with a different constant (C) region gene such as IgG. In the case ofprotein antigens, Th2 cells are thought to be required for switchingfrom IgM to IgG, IgA, or IgE isotypes. IgM is therefore the principalantibody produced during a primary immunization. This primary antibodyresponse is manifested by serum IgM antibodies as early as 3-5 daysafter the first exposure to an immunogen (immunizing antigen), peaks in10 days, and persists for some weeks. Secondary or anamnestic antibodyresponses following repeated exposures to the same antigen appear morerapidly, are of longer duration, have higher affinity, and principallyare IgG molecules.

When antibodies bind to antigens, they may 1) neutralize pathogenicfeatures of antigens such as their toxins, 2) facilitate their ingestionby phagocytic cells (opsonization), 3) fix to and activate complementmolecules to produce opsonins and chemoattractants (vide infra), or 4)participate in antibody-dependent cellular cytotoxicity (ADCC). Inaddition to antibody formation, B cells also process and present proteinantigens. After the antigen is internalized it is digested intofragments, some of which are complexed with MHC class II molecules andthen presented on the cell surface to CD4+ T cells.

In the case of aquatic vertebrates, examples of pathogenicmicroorganisms whose antigenic determinants may be expressed in thetransgenic algae include, but are not limited to Rennibacteriumsalmoninarum (causative agent of bacterial kidney disease in salmon,trout, char and whitefish; i.e., salmonids), Aeromonas salmonicida,Aeromonas hydrophila, species of Vibrio (including V. anguillarum and V.ordalii), species of Pasteurella (including P. piscicida), species ofYersinia, species of Streptococcus, Edwardsiella tarda and Edwardsiellaictaluria; the viruses causing viral hemorrhagic septicemia, infectiouspancreatic necrosis, viremia of carp, infectious hematopoietic necrosisvirus, channel catfish virus, grass carp hemorrhagic virus, nodaviridaesuch as nervous necrosis virus or striped jack nervous necrosis virus,infectious salmon anaemia virus; and the parasites Ceratomyxa shasta,Ichthyophthirius multifillius, Cryptobia salmositica, Lepeophtheirussalmonis, Tetrahymena species, Trichodina species and Epistylus species.

Examples of proteins for inducing an immune response in certain aquaticvertebrates include but are not limited to p57 leukocyte agglutinizingprotein from Rennibacterium salmoninarum and the G-protein frominfectious hematopoietic necrosis virus (IHNV).

Aquatic invertebrates which are suitable host animals include, but arenot limited to, shrimp, crabs, oysters, and clams.

Shellfish possess a defense system capable of defending againstpathogens. This system has many similarities to the nonspecific defensesystem of vertebrates, such as the activation of phagocytotic cells(hematocytes). However, there is no evidence that shrimp or otherinvertebrates possess a specific defense response similar to the immunesystem of vertebrates. Other defense responses activated followinginfection reported for shrimp include: increases in hematocyteproduction, production of active oxygen species, activation ofphenoloxidase and subsequent melanin synthesis, the production ofantimicrobial peptides, encapsulation of the foreign body andcoagulation of hematocytes (Rodriquez, et al. 2000, Aquaculture172:351-372). Unique to invertebrates are specific proteins, such as abeta glucan-binding protein (BGBP) and a lipopolysacchamide-bindingagglutinin (LSBA), that recognize cell-wall components of microorganismsand subsequently mobilize hematocytes for phagocytosis (Bachere, E.,2000, Aquiculture 191: 3-11).

Invertebrates have many similarities to the defense response in plants,such as the production of phenolic compounds and reactive oxygen speciesand the mechanism in which the defense system is activated. Theplant-pathogen interaction is a well-characterized signal transductionsystem composed of nonspecific and specific pathogen elicitors—many theproduct of an avirulence gene (avr)—and their cognate plant receptors.Following receptor recognition of an elicitor, intracellular mediatorsorchestrate a cascade response that activates several well-characterizeddefense mechanisms and, is some cases, short-term acquired immunitymediated by systemically transported signaling molecules. BGBP and LSBAhave an analogous role in orchestrating a defense cascade in shrimp thatalso includes a short-term acquired immunity.

Numerous signaling molecules can activate the defense system ofinvertebrates, most of which are of pathogen origin. Treatment with deadbacteria or cell wall components, such as B-glucans,lippopolysaccarides, and peptoglycans, have been reported to provideprotection against shrimp pathogens when administered prior tochallenge. (Alabi, et al. 1999, Aquaculture 178:1-11) reported thatfresh or freeze dried Vibrio harveyi administered by immersion, but notorally, reduced infection of P. indicus protozoa by V. harveyi for 48hours. More virulent strains increased the level of protectionsuggesting that the defense response is activated either by differentdefense eliciting molecules (qualitative) or different numbers of thesame defense eliciting molecules (quantitative). Furthermore, testedstrains provided cross-protection suggesting that this is a non-specificresponse. Others have reported that peptoglycan extracted from the cellwall of the nonpathogen Bifidobacterium thermophilium, providedprotection against vibrosis and white spot syndrome baculovirus whenadministered orally. These results further support that invertebratesrespond to pathogens through a general nonspecific mechanism that can beactivated by different elicitor molecules and is effective against abroad range of pathogens. These defense-activating signaling molecules(elicitors) do not result in the production of antibodies. However, forpurposes of convenience, the term “antigenic determinant” as used hereinalso encompasses the elicitors which prompt a defense response inaquatic invertebrates.

Examples of microorganisms which are pathogenic for aquaticinvertebrates include, but are not limited to, white spot syndrome virus(WSSV), tuarus virus, IHHNV and Vibrio harveyii.

Examples of proteins for inducing an immune response in invertebratesinclude, but are not limited to, the VP28, VP26c and VP24 proteins ofWSSV shrimp virus. See van Hulten et al., Virology 266:227-236 (2000)and WO 0109340 for a discussion of WSSV and its use in vaccines forcrustaceans.

Algae

Algae are plant-like organisms without roots, stems or leaves. Algae aredistinct from plants in several ways, including phylogenetically,biochemically, and morphologically. Algae contain chlorophyll and varyin size from microscopic forms (phytoplankton) to massive seaweeds.Their habitat is fresh or salt water, or moist places. Most algae areeukaryotic (sub-kingdom=phyciobionta), but several (e.g., cyanobacteriaand prochlorophyta) are prokaryotic.

The transgenic algae of the present invention encompass both prokaryoticand eukaryotic algae, which preferably are unicellular. Unicellularalgae are also known as microalgae. Preferably, the present transgenicof the present invention algae comprise walls that either lack pores orgaps (referred to hereinafter as “walled” algae) or that contain poresor gaps (referred to hereinafter as “wall-less” algae). Optionally, thewall-less algae are coated with a polysaccharide polymer as described in(Tsai, et al., 1994, Progress. Fish-Culturist 56: 7-12)

The transgenic algae of the present invention comprise a transgenecomprising an exogenous polynucleotide which encodes the antigenicdeterminant and is operably linked to a promoter which drives expressionof the exogenous polynucleotide in the algae, and preferably comprisesother genetic elements required for expression. Preferably, thetransgene comprises a terminator for ending transcription. Alsopreferably, the transgene is stably integrated into the genomic materialfound in the nucleus (known hereinafter as a nuclear transformant), thechloroplast (known herein after as a chloroplast transformant), ormitochondria (known hereinafter as a mitochondrial transformant’) of thealgae. In nuclear transformants, the antigenic determinants preferablyare expressed on the cell membrane or cell wall of the transgenic algaeor in the cytoplasm thereof.

In certain preferred embodiments, the algae is a green algae(Chlorophyta), a brown algae ((Phaeophyta), or diatoms(Bacillariophyta).

Examples of green algae, which are especially well-suited for use as thedelivery system, include members of the Chlamydomonas species,particularly Chlamydomonas reinhardtii; the Chlorella species, theVolvox species, and some marine macrophytes. Chlamydomonas reinhardtii,a unicellular eukaryotic green algae is particularly advantageous foruse in introducing antigens into animals. C. reinhardtii growvegetatively through mitotic division of haploid cells. Haploid cellsare of either the (−) or (+) mating type. When grown in the absence ofnitrogen, haploid cells of opposite mating types associate, are heldtogether through their flagella, and eventually fuse to form a diploidzygospore. The diploid zygote undergoes meiosis and releases fourhaploid cells that resume the vegetative life cycle. One example of awalled green algae is Chlamydomonas strain CC-744. One example of awall-less green algae is Chlamydomonas strain CC-425. Both of thesestrains are available from Chlamydomonas Genetic Stock Center, DukeUniversity (Durham, N.C.).

Chlamydomonas reinhardtii is particularly preferred because it growsrapidly and is easily and inexpensively grown in culture. Exogenous DNAcan easily be introduced into the nuclear, chloroplast, andmitochondrial genome of this algae, and can be expressed at highefficiency (≧1% of total cellular protein). Auxotrophic mutants (mutantsthat differ from the wild-type in requiring one or more nutritionalsupplements for growth) are readily available at the ChlamydomonasGenetic Stock Center. Such mutants can be genetically complemented bytransformed DNA (i.e., exogenous DNA introduced into the cell), whichfacilitates selection of algae containing a desired transgene. Thisselection method is preferred, at it is free from use of antibiotics.

In addition to the ease of growth and genetic manipulation, as citedabove, there are additional characteristics of C. reinhardtii that makethe organism useful for delivering antigens to animals. C. reinhardtiiis a potential food source for animals, especially larval fish andmarine invertebrates (C. reinhardtii is nontoxic and nonpathogenic. Bothfreshwater C. reinhardtii and a related marine species, C. pulsatilla,are available for administering antigens to aquatic organisms in bothenvironments.

Optionally, the algae of the present invention are geneticallyengineered such that they will not proliferate unless they are in veryspecific controlled environments (i.e., such strains will not grow ortransfer their genes in the wild). Within the context of thisapplication, such algae are said to be “disabled.” Use of such disabledstrains inhibits or limits spread of the transgenic algae of the presentinvention into the environment.

Such disabled strains of algae, particularly strains of C. reinhardtii,are constructed by incorporating into the genomes of such strainsvarious genetic mutations that preclude growth and/or mating outside ofa specific environment. For example, the transgenic algae may beengineered to contain mutations that prevent photosynthesis. Strainscontaining such mutations are unlikely to survive in the wild becausethey cannot produce the energy or reduced carbon necessary to sustainlife. Such mutant-containing strains, however, can be grown in thelaboratory using acetate as a carbon source. One such type of mutationpreventing photosynthesis occurs in, but is not limited to, genescomprising the psbD/psaC operon of C. reinhardtii, which is part of thechloroplast genome of the organism. Such mutations in the chloroplastgenome are preferably in the chloroplast genome of cells of the (+)mating type. C. reinhardtii of the (−) mating type generally do notsurvive after a mating to produce a diploid cell has occurred (seebelow).

Other mutations useful in constructing disabled algal strains aremutations in genes resulting in strains that cannot grow in the absenceof specific metabolites (i.e., substances produced by, or taking partin, metabolism). Such strains are said to be “auxotrophic” for thatparticular metabolite. Strains auxotrophic for various amino acids,vitamins, nucleotides, and so forth are particularly useful. Forexample, some such useful mutations require cells to be grown in thepresence of arginine, thiamine, or nicotinamide.

Other mutations that affect the ability of algae to grow and/or transferits genes, although such mutations are not specifically stated herein,are also included within the scope of this invention.

In one embodiment of this invention multiple mutations of the typedescribed above, for example, are combined into a single strain ofalgae. Combinations of these mutations in a single strain (also called“stacking” of mutations) result in disabled strains that areparticularly nonfunctional in growth and mating. Such strains of algaeare particularly unable to grow and transfer their genes in the wild.

Another strategy useful in making disabled algal strains embodied inthis invention takes advantage of events that occur naturally in amating event. In C. reinhardtii, when haploid (−) and (+) cells mate toform a diploid cell, only the chloroplast genomes from the (+) matingtype organism survive. The chloroplast genomes of (−) mating type cellsare degraded during mating. Therefore, C. reinhardtii strains in whichthe transgenes encoding the antigenic determinant are located in thechloroplast genome of a (−) cell are advantageous when control ofproliferation of transgenic algae is desired.

Another type of disabled algal strain that is included in this inventionare algal strains that have mutations in the genes encoding flagella.For example, in C. reinhardtii, flagella are necessary to hold (−) and(+) cells together so that mating can occur. Therefore, certaintransgene-containing cells with mutations in genes encoding flagellawill be unable to transmit the transgene through mating.

An additional disabling strategy that is part of the present inventionis use of the freshwater alga, C. reinhardtii, for use in transferringantigens into saltwater aquatic organisms, as C. reinhardtii is unableto survive in seawater for more than an hour. C. reinhardti strainscontaining the P5CS gene for proline synthesis can also be usedsimilarly when survival of the algae for longer times is important for aparticular application. Strains containing the P5CS gene can tolerateseawater for up to 10 hours before 100% mortality occurs.

Preparation of the Transgenic Algae

In addition to an exogenous polynucleotide encoding an antigenicdeterminant of a pathogenic microorganism, the transgene which isincorporated into the transgenic algae comprises a promoter whichregulates transcription of the exogenous gene in the nucleus,chloroplast, or mitochondria, of the algae, and preferably other geneticelements required for expression. The transgene also preferably includesa terminator for terminating transcription.

To prepare vectors for making the transgenic algae, the exogenouspolynucleotide encoding the antigenic determinant is first cloned intoan expression vector, a plasmid that can integrate into the algalgenome. In such an expression vector, the DNA sequence which encodes theantigenic determinant or a fusion protein comprising the antigenicdeterminant is operatively linked to an expression control sequence,i.e., a promoter, which directs mRNA synthesis. Preferably, the promoteris an endogenous promoter, i.e., it directs transcription of genes thatare normally present in the algae. Examples of suitable promoters forChlamydomonas reinhardtii include, but are not limited to, thechloroplast gene promoter psbA and the nuclear promoter region of the β₂tubulin gene. The expression vector, may also contains a ribosomebinding site for translation initiation and a transcription terminator.Preferably, the recombinant expression vector also includes an E. coliorigin of replication and an E. coli selectable marker to facilitatecloning of the vector in E. coli.

In one embodiment, the exogenous polynucleotide encoding the antigenicdeterminant is fused to a polynucleotide which encodes a membraneprotein to express the antigen on the surface of the cell. For example,the predicted folding topology of the CO₂-induced surface protein ofChlamydomonas reinhardtii indicates that both the N-terminus and theC-terminus are located on the periplasmic surface of the plasmamembrane. Thus, this protein is especially useful for expressing theantigenic determinant on the plasma membrane of Chlamydomonasreinhardti.

Plasmids are introduced into the algae by standard transformationmethods known to those skilled in the art, such as for example,electroporation, vortexing cells in the presence of exogenous DNA, acidwashed beads, polyethylene glycol, and biolistics.

One particular advantage of the present invention is that genes formultiple antigens from one infections agent, or multiple antigens fromdifferent infectious agents, can be expressed simultaneously in a singlealgal cell. Such multiple genes or epitopes can be included in a singlevector, for example a plasmid, or in multiple plasmids, each of whichmust be transformed into the algae. The advantage of such an algae thatexpresses multiple antigens, is that all of the antigens are introducedinto the animal by administration of the particular algal strain to theanimal.

Transformation of the algae is determined by assaying for the presenceof the gene encoding the antigen by PCR, for example. Procedures knownto those of skill in the art, such as for example, deflagellation,copper addition, and ammonium addition of the algae, may be used toenhance expression of the antigenic determinant in the transgenic algae.The choice of such procedure depends upon the promoter used to preparethe construct. See, for example, Davies et al., Nucleic Acids Research20: 2959-2865 (1992); Dutcher, Current Opinions in Cell Biology 13:39-54(2001); Moseley et al., Photosynthesis: Mechanisms and Effects.

One type of plasmid vector integrates into the nucleus of algal cellsand expresses its proteins which are localized to the cytoplasm of algalcells. One particular vector of this type is pSSCR7, derived from a theplasmid described in Davies (Davies et al. (1994) Plant Cell 6:53-63).

Another type of vector also integrates into the nucleus but expressesproteins that are localized to the periplasm. One particular vector ofthis type is a derivative of pSS CR7 which has a 5′ aryl sulfataseperiplasmic targeting transit sequence (Davies et al. (1994) Plant Cell6:53-63).

A third type of vector integrates into the chloroplast genome byhomologous recombination and expresses proteins that are localized tothe chloroplasts (Hutchinson, et al., 1996, Chapter 9, Chloroplasttransformation. Pgs. 180-196; In: Molecular Genetics of Photosynthesis,Frontiers in Molecular Biology. Anderson B., Salter AH, and Barber J.eds.: Oxford University Press).

Administration of Transgenic Algae to Animals

Animals to which the transgenic algae are administered include, but arenot limited to, mammals, birds, and aquaculture species. Aquaculturespecies include a diversity of species of cultured fin-fish, shellfish,and other aquatic animals. Fin-fish include all vertebrate fish, whichmay be bony or cartilaginous fish. Such fin-fish include but are notlimited to salmonids, carp, catfish, yellowtail, seabream, and seabass.Salmonids are a family of fin-fish which include trout (includingrainbow trout), salmon, and Arctic char. Examples of shellfish include,but are not limited to, clams, lobster, shrimp, crab, and oysters. Othercultured aquatic animals include, but are not limited to eels, squid,and octopi.

One method of administering the present transgenic algae to animals isoral delivery of the algae to the animal by feeding. The transgenicalgae may be delivered to the animal as a dried cell powder or as acomponent of the normal diet. For example, in one method of feeding thealgae to fish, up to 5% freeze-dried transgenic algae are added to anaqueous mixture containing 5% of a casein-gelatin based protein source.The ingredients are cold-pelleted, freeze-dried, crushed into 2 mmparticles, and fed to the fish. Live algae could be delivered in gelatincapsules.

Another method of administering algae to animals, particularly aquaticanimals, is immersion of the host animal in a suspension of live algae.Good results have been obtained by immersing trout in an aqueoussuspension containing from between 10⁵-10⁷ algae per ml of water. Fishwere immersed in the suspension anywhere from between 20 seconds to 2hours, and then removed from the suspension. The immersion method isparticularly advantageous for introducing algae into smaller fish (lessthan about 10 to 15 grams in weight). Such method can be used incombination with oral delivery of algae though feeding, as describedabove. Other methods of delivery are possible and are within the scopeof this invention.

The transgenic algae are introduced into the animal in a regimendetermined as appropriate by a person skilled in the art. For example,the transgenic algae may be introduced into the animal multiple times(e.g., two to five times) at an appropriate interval (e.g., every two tothree weeks) and dosage or dilution, by normal feeding or by immersion.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the invention as defined in the claimswhich are appended hereto. The references cited in this document arespecifically incorporated herein by reference.

Example 1 Transgenic Algae Expressing the P57 Immunogen fromRennibacterium salmoninarum

Rennibacterium salmoninarum is the etiologic agent for bacterial kidneydisease, the most common disease of farmed salmonoids. A transgenicalgae expressing an antigenic determinant of the fish pathogen. R.salmoninarum was prepared using a portion of the P57 leucocyteagglutinizing protein (Genbank accession no. AF123889) of Rennibacteriumsalmoninarum for surface display as an antigen.

A highly antigenic determinant of the P57 protein encoding the aminoacids VYNKDGPAKELKV, (SEQ ID NO: 1) residues 112-124, was identifiedusing the program Sciprotein, Scivision (Burlington, Mass.). Thefollowing is a synthetic oligonucleotide encoding this peptide and usingthe codon bias preferred for expression of nuclear genes in the algaChlamydomonas reinhardtii:

(SEQ ID NO. 2) 5′-GATCTAGATTAACCTTCAGCTCCTTGGCGGGGCCGTCCTTGTTGT   ACACGCCCCCACCTTGGTGCGCCGTCAGAG-3′The above synthetic oligonucleotide was used to generate a fusion genebetween the 3′ end of a low CO₂-induced plasma-membrane protein gene(Genbank accession no. U31976) and the P57 antigen encoding sequencecreating by adding the above P57 fragment 3′ of nucleotide 677 ofU31976, shown below:

(SEQ ID NO.: 3) 5′-ATGTCGGGCT TGAACAAGTT CATCTATGTG GGCCTCGTTA   TCTCGCAGCT GCTGACTCTG GCGGCCTACG  TGGTCGTCAC   GGCCGGCGCT GCCCTTCTGC AGAAGAAGGC GAACACGCTC   ACTCTGTTTG ACACCCAGGA GGGCATTGAC AAGTACACTC   CCGTTTACAA GGAGGTCTTC ACGGCGACCA CCTACATCAT   CGCCTACCCC CAGCAGCCCC AGTACCAGTT CCAGTACCAG   TGGTGGATCA TCCAGTTCGA GCTGTTTGTG TTCCTGCTGA   CCGCCGCCTG CACCGTCTTC CCCTCCATCA TCAAGCGCAT   GCGCCCCGTG GCCCTGACCT TCATCGCCTC CGCCCTGGTG   CTGGTCATGG ACAACATCAA CGCCATCTTC TTCCTGCTCC   GCAACGAGAC CGCCACCGCT GTGTTCGACG ACTACCGCAT   CGCCACCGCT CAGGCTGGCC TGATCATGGT TGGCGTGGCG   AACGGCCTGA CCATCTTCTT CCTGGGCTCG TACGACGCTG   AGGAGTCGCA TGCGATGCCC AACGTGCACG TCACCTCTGA   CGGCGCCACC AAGGTGGGCG GCGTGTACAA CAAGGACGGC   CCCGCCAAGG AGCTGAAGGT GTAA-3′It was expected that the P57 epitope would be expressed on the periplasmside of the cell membrane, as shown in FIG. 3. The gene fusion then wascloned into the multi-cloning site of plasmid pSSCR7 under control ofthe β₂ tubulin promoter of Chlamydomonas. pSSCR7 was constructed bycloning the HindIII/EcoRI fragment (˜2.7 Kbp) that carries theChlamydomonas β ₂-tubulin promoter and the 5′ end of arysulfatase gene(˜1.0 Kbp) from pJD55 (Davies, 1992 Nucleic Acids Research 20:2959-2965) into pUC18, and designated as pβ₂TU1. In order to eliminatethe 5′ end of arysulfatase gene, the β₂-tubulin promoter was amplifiedby PCR. The PCR product was cloned into the BamHI/EcoRI sites of pUC18to make plasmid pβ₂TU2. The TATA box was found by DNA sequence analysis˜100 bp away from BamHI site. In order to introduce a unique NdeI sitewhich contains an ATG codon, and a unique NarI site, which was used toclone the 3′ terminator, a NdeI site was removed from pUC18 to make pUNand both sites were removed from pUC18 to make pUNN. A XhoI/Nar fragmentcontaining the 3′-terminator from Chlamydomonas low CO₂-induced membraneprotein gene, as follows:

(SEQ ID NO. 4) 5′-GGCGCCATCT AAGCAGAAGG CTGTGGGATG TGTCACCGTT   AAGCATCGGA GTTTGGGAAG TAGAGAATCT GGGGCTGCGG   TTTTGTGGTT TGCCGCTGCG GTCTGCACTT GGCAGGGTTG   CCCCAGGTCT TGGGGTGACA GTTTAGTTGC TAGGTTGGTA   GCATGTCCTT CGTGACACCA GCGCATTGCA CCCGCTATGT   ACATTCATCG TTTTGGGTCT GGAGCGCTGC GCAGCACCTT   TGGGTAGCGA ATACTTCGGG TGAGCTGCTT ATCTGTATGG   TACGGATGGG CACGGCTCCA AGCAGCAATA CACGGACGCA   CATGCACCAA ATTTTGGTTG TTTGAGTGGA CCGGCTTTAT   CCAACGGTTC AGGTTTGGTT GCTCTCTCCA TCGGAAGCAG   AGCAGAAGCA CAACACACGT CGCAAACATG ATTGGAGCCA   AGGAGCATGA AATGCGAAAG AGCTGGACCA TGCACAGCGC   ATGTAATAAG AGTACTGCAG A-3′was combined with β₂-tubulin promoter to form expression vector pSSCR1.A KpnI/SstI fragment containing the multicloning sites from pBluscriptII KS was cloned into pSSCR5 to make the final expression vector,pSSCR7. (FIG. 1.)

As seen in FIG. 1, the pSSCR7 has sixteen useful cloning sites includingNaeI, KpnI, ApaI, XhoI, SalI, ClaI, HindIII, EcoRV, EcoRI, PstI, SmaIBamHI, SpeI, XbaI, NotI and NarI, that can be used for introducing oneor more coding sequences, as shown in FIG. 1. The fusion gene describedabove was cloned into the NdeI and XbaI cites.

The resulting plasmid (pCREpitope) was co-transformed into the nucleusby electroporation into Chlamydomonas reinhardtii strain CC-425 using anequimolar amount of p389, a plasmid containing the Arg-7 gene (seeDebuchy et al., EMBO, 1989. Vol 8, 2803-2809). The Arg-7 genecompliments the arginine auxotrophic strain of Chlamydomonas, CC-425(Debuchy et al., EMBO, 1989. Vol 8, 2803-2809). Transformants wereobtained after plating the cells on TAP-agar containing 100 μg arginineper ml of medium. The plates were illuminated with fluorescent tubes at10 mmol photons/m²/sec at 22-27° C. Transformants were found after 9-10days of incubation. The resulting transfected algae expressing thefusion protein are known as E-22.

The main features of the new expression vector are (i) the use of theβ₂-tubulin promoter, (ii) the construction of sixteen unique cloningsites, (iii) the use of high copy number (in E. coli), pUC18, as theoriginal plasmid, and (iv) the small size of new expression vector (4.4Kpb).

Another plasmid was created by inserting the entire P57 gene intopCPPTG, as shown in FIG. 2. pCPPTG is a plasmid constructed forexpression of foreign genes in chloroplasts. It based on a modified E.coli vector, pUC18. As seen in FIG. 2, pCPPTG comprises the psbApromoter, psbA terminator, aadA cassette, and a multicloning sitelocated between the psbA promoter and terminator. The psbA promoter,psbA terminator, and aadA cassette are derived from the pBA155dH3plasmid, while the multicloning site is derived from pBluescript IIKS(described in Hutchison et al, (1996) Chapter 9, and Ruffle et al,Chapter 16; In Molecular Genetics of Photosynthesis, Frontiers inMolecular Biology. Oxford Univ. Press). The entire P57 gene from R.salmoninarum was cloned into the multicloning site, specifically theApaI and PmeI sites, of pCPPTG.

Co-transformants growing on medium lacking arginine and containing themembrane protein-P57 fusion were identified by PCR amplification of themembrane protein-P57 fusion gene using the following oligonucleotideprimers:

(SEQ ID NO. 5) Primer C113-5′-AGCATATGGGGCCCATGTCGGGCTTGAACAAGTT               CATCT (SEQ ID NO. 6)EPITOPE-3′-GATCTAGATTAACCTTCAGCTCCTTGGCGGGGCCGTC           CTTGTTGTACACGCCCCCACCTTGGTGCGCCGTCAGAG

In order to perform PCR on the transformants, total genomic DNA from C.reinhardtii was isolated. To do this, cell cultures (20 ml) werepelleted by centrifugation and resuspended in 0.35 ml TEN buffer. Theresuspended cells were incubated with 50 μl of 2 mg/ml proteinase K and25 μl of 20% SDS for 2 hr at 55° C. Then, 50 μl of 5M potassium acetatewas added and the cells were incubated on ice for 30 min. The lysate wasextracted by phenol:chloroform and DNA was precipitated by ethanol.

Those cells that were positive for the presence of the membraneprotein-P57 fusion product by PCR were then were screened by westernblot analysis using antibodies against the intact P57 protein. Asdemonstrated by western blot analysis (see FIG. 5), antibodies generatedagainst the intact P57 protein recognized the 57 kD P57 protein fromsolubilized whole cell extracts of transgenic algae (CP57) expressingthe P57 protein. No protein was detected on the western blot fornon-transformed cells.

Example 2 Immersion

Transgenic Chlamydomonas reinhardtii expressing the P57 protein fromRennibacterium salmoninarum as a fusion protein on the plasma membrane(called E-22 algae) or in the chloroplast (called CP57 algae) wereconstructed as described in Example 1. Control algae (the CC-2137 strainof C. reinhardtii) were also used to test administration by immersion.

The algae were grown in tris-acetate-phosphate (TAP) medium. See GormanDS and Levine RP (1965), Proc. Nat. Acad. Sci. 54: 1665-1669, to adensity of 1×10⁶ cells/ml at 22-27° C. with 10 μmol photons/m²/secillumination from fluorescent tubes. The algal cells were harvested bycentrifugation and resuspended in water to a density of approximately1×10⁶ cells/ml.

Rainbow trout juveniles (average initial weight, 9.1±0.5 g) weresubjected to a bath treatment in water containing algae in a 20 L barrelcontainer (one barrel containing each of the E-22, CP57 and CC-744algae). Exposure of the trout to the various algae was for 2 hours withintense aeration of the water. The concentrations of the three types ofalgae were as follows: CP57, 7.97×10⁵ cells/ml; 2137, 2.71×10⁶ cells/ml;and E-22, 2.97' 10⁶ cells/ml. A control treatment was also performedwhere additional fish were handled identically, except that they werenot exposed to algae (sham immersion). After immersion, both controlsand those immersed in algae, were distributed into 3 tanks per immersiontreatment. All the fish groups were fed with the same commercial diet atthe rate of 2% of fish weight for 3 weeks (Bioproducts, Inc. Oregon).After that period, the fish were immersed again in a 2^(nd) treatmentaccording to the same procedure as in the first treatment. Theconcentrations of the three algae types for the 2^(nd) treatment were asfollows: CP57, 1.46×10⁶ cells/ml; 2137, 6.57×10⁵ cells/ml; and E-22,1.23×10⁶ cells/ml. The fish were fed with the commercial diet for 4weeks until the second sampling in the experiment (7 weeks) (a firstsampling was performed prior to any feeding). All fish were fasted for24 h prior to treatment.

The water temperature increased gradually from 12 to 17° C. during thecourse experiment due to seasonal environmental changes. Diurnallight:dark cycle was regulated at 12 h:12 h. Total fish weight in eachtank was measured 3 weeks after the first treatment, and then at 7 and 9weeks after the first immunization. The fish were kept for the last 2weeks at the feeding rate of 1.5% of fish weight.

Blood was taken before the initiation of the experiment and at the timeof weighing fish (3, 7, and 9 weeks) from caudal vein with heparinizedsyringe for plasma, and with non-heparinized syringe for serum. Six fishwere randomly selected per tank for the determinations of hematocrit,hemoglobin, liver and spleen weights. Mucus from the fish was collectedand frozen. Hematocrit was determined by the microhematocrit method.Total hemoglobin was determined with Sigma Diagnostic Kits (procedureNo. 525) by using human hemoglobin solution as standard. All proceduresand handling of animals were conducted in compliance with the guidelinesof the Institutional Laboratory Animal Care and Use Committee, The OhioState University.

TABLE 1 Weight, Spleen Relative Weight (SRW), Hematocrit and Hemoglobinof Fish Treated by Immersion Final weight SRW¹ Hematocrit HemoglobinTreatment (mean ± SD) (% body weight) (%) (g/100 mlk) Control 22.0 ±1.90  0.080 ± 0.015 ^(ab) 37.8 ± 3.51 8.47 ± 1.09 E-22 22.4 ± 1.36 0.082± 0.012 ^(a) 36.2 ± 2.57 8.39 ± 0.85 CP57 21.5 ± 1.14 0.097 ± 0.004 ^(a)36.5 ± 1.50 7.66 ± 0.31 2137 21.8 ± 1.34 0.082 ± 0.004 ^(a) 35.2 ± 2.257.84 ± 0.77 ¹Spleen relative weight (spleen wt. × 100/body wt) wasmeasured after 9 weeks. All other values were measured after 7 weeks. ²Values (±SD) having different superscripts are significantly different(P < 0.05)

The fish growth and physiological parameter values in the table aboveshow that there were no significant differences in growth rate (finalweight), hematocrit, and hemoglobin among all the treatment groups(P>0.05).

Example 3 Feed Pellet

Transgenic Chlamydomonas reinhardtii expressing the P57 protein fromRennibacterium salmoninarum as a fusion protein on the plasma membrane(called E-22 algae) or in the chloroplast (called CP57 algae) wereprepared as described in Example 1.

The algae were grown in TAP medium as described above in Example 2 to adensity of 1×10⁶ cells/ml. The algal cells were harvested bycentrifugation, frozen in liquid nitrogen and freeze-dried.

Three semi-purified diets formulated based on casein-gelatin as aprotein source were used for oral administration. The threesemi-purified diets were isonitrogenous and isocaloric to incorporate 4%(on dry weight basis) of three different types of algae. The three algaewere E-22, CP57, and CC-2137. Five percent of fish protein concentrate(CPSP 90, Sopropeche S. A., Boulogne-Sur-Mer, France) was supplementedinto the diets to enhance their palatability. The dietary ingredientswere mixed with distilled water and cold-pelleted into 2.0 mm diametersize, and then freeze-dried to have less than 5% moisture. Diets werecrushed and sieved into a desirable particle size (0.8-2.0 mm), andstored at −20° C. until use.

Rainbow trout juveniles (average initial weight, 9.1±0.5 g) wererandomly distributed into 24 rectangular tanks (20 L capacity) at adensity of 22 fish per tank, 3 tanks per dietary treatment. A controlgroup was also used (each in triplicate tanks) and were fed a commercialdiet (Bioproducts, Inc. Oregon). The control group was not exposed toany disturbances. Each experimental diet was fed to a group (3 tanks) offish at the feeding rate of 2% of fish body weight for 3 consecutivedays. Accordingly, the results in an approximate intake of 25 mg ofalgae protein/100 g fish body weight/day. After 3 days of feeding algaediets twice a day, all the fish groups were fed with the same commercialdiet for 3 weeks (2.5% per day), twice per day, 7 days per week. The2^(nd) oral treatment (boost) was conducted again with the same algaediets and the commercial diet at the feeding rate of 2% of fish bodyweight for 4 days. After the 4 days of the 2^(nd) oral treatment, allthe fish groups were fed the same commercial diet for 4 weeks until thesecond sampling in this experiment. The fish were kept for 2 additionalweeks at the feeding rate of 1.5% of fish weight and sampled again(third sampling; 9 weeks).

TABLE 2 Weight, Spleen Relative Weight (SRW), Hematocrit and Hemoglobinof Fish Treated Orally Final weight SRW¹ Hematocrit Hemoglobin Treatment(mean ± SD) (% body weight) (%) (g/100 mlk) Control 19.9 ± 0.60 0.093 ±0.004 ^(a) 38.3 ± 5.69 9.52 ± 0.96 E-22 21.5 ± 1.00 0.053 ± 0.001 ^(c)38.5 ± 1.80 8.40 ± 0.83 CP57 22.0 ± 0.12  0.056 ± 0.004 ^(bc) 39.5 ±3.12 8.34 ± 0.62 2137 20.6 ± 0.66 0.052 ± 0.003 ^(c) 35.3 ± 0.58 7.61 ±0.02 ¹Spleen relative weight (spleen wt. × 100/body wt) was measuredafter 9 weeks. All other values were measured after 7 weeks. ² Values(±SD) having different superscripts are significantly different (P <0.05)

The fish growth and physiological parameter values in the able aboveshow that there were no significant differences in growth rate (finalweight), hematocrit, and hemoglobin among all the treatment groups(P>0.05). Spleen relative weight (SRW) was significantly lower in orallytreated fish groups than in a control fish group and all the fish groupsin immersion treatments (P<0.05).

Example 4 Serum and Mucus Antibodies in Fish after Administration ofAlgae

Serum and mucus obtained from the fish treated by the immersion method(Example 2) as well as the oral feeding method (Example 3) were examinedfor the presence of antibodies reactive with the P57 immunogensexpressed by the transgenic algae. This was done by using the serum orthe mucus from fish fed CP57, E-22, CC-2137, or no algae to probewestern blot membranes to which were transferred proteins from SDS-PAGEgels of solubilized wild-type Chlamydomonas cells or transgenicChlamydomonas expressing the P57 protein in the chloroplast (CP57), oras a fusion protein between the high CO₂ induced protein and the P57antigenic determinant (E-22).

Wild-type, E-22 and CP57 algae were boiled in SDS-PAGE loading bufferfor 5 minutes. Samples were loaded at 15 μg chlorophyll per well (seeArnon D (1949), Plant Physiol. 24: 1-15, for additional information onchlorophyll assays) on a 12.5% acrylamide gel with a 6% acrylamidestacking gel. The samples were electrophoresed at 15-18 mAmp constantcurrent for 5-6 hr. Following electrophoresis the proteins weretransferred from the gel to immobilon-P (PVDF=polyvinylidene fluoride)membrane using a semi-dry system at 1.25 mA per square centimeter ofmembrane for 2-3 hr. After removing the membrane from semi-dryelectroblotter, the membrane was soaked in methanol for 15 second anddried at room temperature for 30 minutes. The membrane was then washedtwice with PBST buffer (10 mM Sodium phosphate, 150 mM NaCl, 1%Tween-20, pH 7.4) for 5 minute each. (0.25 mL PBST per square centimeterof membrane) and blocked with 3% casein (in PBST buffer) at 0.25 mL persquare centimeter for 1-2 hr. The membrane was then washed twice withPBST buffer (10 mM Sodium phosphate, 150 mM NaCl, 1% Tween-20, pH 7.4)for 5 minute each. (0.25 mL PBST per square centimeter membrane).

All fish mucus and serum were treated with soluble protein from CC-425algal strain at 1:2 (vol/vol ratio) for 1 hr at room temperaturefollowed by centrifugation to remove non-specifically bound proteins.Fish mucus or sera was used at a 1:150 dilution in 1% BSA in PBST andincubated at room temperature for 2 hr (used 0.1 mL of serum or mucussolution in buffer per square centimeter of blotting membrane). Themembrane was then washed twice with PBST buffer (10 mM sodium phosphate,150 mM NaCl, 1% Tween-20, pH 7.4) for 5 minute each (0.25 mL PBST persquare centimeter of membrane). This was followed by incubation withmouse anti IgM Rainbow trout serum (1:200 dilution in 1% bovine serumalbumin (BSA) in PBST) at room temperature for 2 hr (0.1 mL dilutionbuffer per square centimeter of membrane). The membrane was then washedtwice with PBST buffer for 5 minute each (0.25 mL PBST per squarecentimeter of membrane) followed by incubation with goat anti-mouseantisera conjugated to horseradish peroxidase (BRP) at a 1:3,000dilution in 1% BSA in PBST at room temperature for 1 hr (0.25 mLdilution buffer per square centimeter of membrane). The membrane wasthen washed twice with PBST buffer for 5 minute each. (0.25 mL PBST persquare centimeter of membrane). The HRP detection system was Opti-4CNSubstrate Kit from Bio-Rad. Opti-4CN is an improved and more sensitiveversion of the colorimetric horseradish peroxidase (BRP) substrate,4-chloro-1-naphthol (4CN). Normally, this step takes time about 20-30minutes (0.25 mL substrate per square centimeter of membrane).

The results of the studies are shown in FIG. 4 which shows sera fromfish that were fed algae in their diet, and in FIG. 5 which shows mucusfrom fish that were immersed in algae.

The first western blot (FIG. 4) shows that sera from fish fed pelletscontaining either E-22 or CP57 algae recognized a 57 kD protein presentin algae (CP57) expressing the P57 protein in the chloroplast (lanes 2and 5). Significantly, no 57 kD band was detected when using sera fromfish fed either wild type or no algae in the diet. In addition, twoprotein bands were detected at 22 and 27 kD in all serum treatments.These bands arise from non-specific interactions. In contrast, no 57 kDproteins were detected in serum from fish that were immersed in eitherE-22, CP57, wild-type or no algae. These results indicate that fish fedfood containing transgenic algae expressing the P57 protein ofRennibacterium salmoninarum generated specific antibodies against theP57 protein. Unfortunately, due the cross-contaminating band at 22 kD itwas not possible to determine whether fish fed pellets containing E-22algae generated antibodies against the 22 kD fusion protein. However,the fact that the P57 protein present in CP57 algae was detected usingsera from E-22 fed fish suggests that the 22 kD fusion protein wasimmunogenic against the P57 antigenic determinant present in the fusionprotein.

In addition, fish mucus was tested for the presence of antibodiesgenerated against the P57 protein. As shown in FIG. 5, western blotsprobed with mucus from fish immersed in E-22 and CP57 algae wasimmunoreactive against the P57 protein present in CP57 algae (lanes 2and 5). Again no immune reaction was observed using mucus from fishimmersed in wild-type or no algae. These results demonstrate thatP57-specific antibodies were expressed in mucus of fish immersed in E-22and/or CP57 algae. There also appears to be an immune reaction specificfor the 22 kD fusion protein when using mucus from fish immersed in E-22algae (lane 1). However, this result is tentative since a non-specificinteraction is observed at the same molecular weight in lanes 5 and 6.Significantly, immersion of fish with any algal strain failed togenerate P57-specific antibodies in sera. Overall, these resultsindicate that immersion with algae expressing foreign and immunogenicproteins (P57) is an effective means to generate the production ofprotective antibodies in the mucus of fish.

Example 5 Algae Expressing WSSV Proteins and Administration to Shrimp

White Spot Syndrome Virus (WSSV) is a cause of disease of shrimp. Shrimpproduction losses of 80% due to WSSV infection have been reported andshrimp farms can be shut down for periods of up to two years following aWSSV infestation.

VP28, VP26, VP24, and VP19 are known proteins from WSSV (PCT Int. Appl.No. WO 0109340). Using gene specific primers, each of the four knownviral proteins is amplified using WSSV DNA as a template (van Hulten, etal., 2001, PCT International Publication WO 0109340). Each of thesegenes is cloned separately and together into a Chlamydomonas expressionvector similar to the methods described in Example 1. For example thepSSCR7 plasmid which drives high-level expression in the cytoplasm, andthe pSSCR7 vector having a 5′ aryl sulfatase periplasmic targetingtransit sequence (Davies et al., 1994, Plant Cell 6: 53-63). Theaforementioned vectors randomly integrate into the Chlamydomonas nucleargenome. In addition, a proprietary chloroplast transformation vectorthat integrates into the chloroplast genome by homologous recombinationcan be used (Hutchison, et al., 1996, Chapter 9, Chloroplasttransformation. Pgs. 180-196; In: Molecular Genetics of Photosynthesis,Frontiers in Molecular Biology. Anderson B., Salter, A H, and Barber J.eds.; Oxford University Press). Each of these vectors contains promotersthat drive high levels of expression in the cytoplasm or chloroplast.Viral protein expression is quantified by western blot analysisnormalized against known loadings of WSSV (see objective 1A4 forquantification of WSSV). For the western blot analysis polyclonalantibodies are generated against WSSV proteins by injection of purifiedand heat-denatured WSSV into rabbits.

The various expression systems are used to determine which pattern ofexpression (periplasmic, cytoplasmic or chloroplast) most effectivelyinduces the shrimp “immune-response” (Bachere, 2000, Aquaculture 191:3-11; Rombout et al., 1985, Cell Tissue Res. 239: 519-30). Whileperiplasmic expressed viral proteins are expected most effective, theyare potentially most vulnerable to digestion cytoplasmic and chloroplastexpressed proteins would be progressively less susceptible to digestion(D'Souza, et al., 1999, Marine Biol. 133: 621-633).

PSF (pathogenic specific free) shrimp larvae are fed either wild-type orVP-expressing microalgae 3-5 days prior to challenge with known titersof WSSV. During the WSSV challenge, the shrimp larvae are fed theappropriate algal strain either as live algae or as dried algae in feedpellets (D'Souza, et al., 1999, Marine Biol. 133: 621-633). Variousconcentrations of algae feed are compared. Relative percent survivalwill be calculated during the weeks following WSSV exposure for alltreatments. As described below, molecular markers specific for shrimpdisease inducible genes will be used in quantitative real time-PCRexperiments to evaluate the “immune” response in shrimp prior to andafter exposure to 1) WSSV VP expressing algae, 2) wild type algae, 3)WSSV VP expressing algae followed by WSSV exposure, 4) WSSV alone, and4) no algae or WSSV. Based on studies with injected WSSV proteins (vanHulten, et al., 2001, PCT International Publication WO 0109340) it isexpected that shrimp fed microalgae expressing WSSV VP proteins will beprotected from WSSV infection.

Example 6 Rabbits

Transgenic algae expressing antigenic proteins (in the chloroplast,cytoplasm, or on the cell surface as fusion proteins) are delivered torabbits as a component of the feed pellets, essentially as describedabove for fish. Alternatively, the algae are delivered as mixed with thedrinking water. The transgenic algae may, for example, express the CS6and Vi antigens of Salmonella typhi.

Although the invention has been described in detail with reference tocertain preferred embodiments, variations and modifications exist withinthe scope and spirit of the invention as described and defined in thefollowing claims.

1. A delivery system for delivering a biologically active protein to a host animal comprising an algal cell transformed by an expression vector, and the expression vector comprising a nucleotide sequence coding for the biologically active protein, operably linked to a promoter.
 2. The delivery system of claim 1, wherein the algal cell is suspended in water for immersing the host animal.
 3. The delivery system of claim 1 wherein the biologically active protein is selected from the group consisting of hormones and antimicrobial peptides.
 4. The delivery system of claim 1 wherein the algal cell is mixed with a sample of feed for the host animal.
 5. The delivery system of claim 1 wherein the animal is selected from the group consisting of mammals, fish, birds, and crustaceans.
 6. The delivery system of claim 1 wherein the biologically active protein is a peptide derived from the group consisting of bactericidal proteins, insecticidal proteins, growth hormones, and antigens.
 7. A delivery system for delivering antigens to a host animal comprising an algal cell transformed by an expression vector, and the expression vector comprising a nucleotide sequence coding for an antigenic determinant.
 8. The delivery system of claim 7, wherein the expression vector further comprises a promoter operably linked to the nucleotide sequence coding for the antigenic determinant.
 9. The delivery system of claim 8, wherein the expression vector further comprises a terminator for terminating transcription.
 10. The delivery system of claim 9, wherein the algal cell expresses the antigenic determinant in an area selected from the group consisting of nucleus, chloroplast, mitochondria, periplasmic space, cell membrane, or cell wall.
 11. The delivery system of claim 7, wherein the algal cell is selected from the group consisting of green algae, brown algae, or diatoms.
 12. The delivery system of claim 7 wherein the algal cell is Chlamydomonas reinhardtii.
 13. The delivery system of claim 7 wherein the antigenic determinant is a holoprotein or protein fragment from a pathogenic organism in the host animal.
 14. The delivery system of claim 13 wherein the antigenic determinant is part of a fusion protein.
 15. The delivery system of claim 7 wherein the algal cell is disabled.
 16. The delivery system of claim 7 wherein the algal cell is packaged as a food product.
 17. The delivery system of claim 16 wherein the food product is a capsule.
 18. The delivery system of claim 7 wherein multiple algal cells are incorporated into a pellet.
 19. The delivery system of claim 18 wherein the algal cells are selected from the group consisting of living and dead cells.
 20. The delivery system of claim 7 wherein the algal cell is suspended in water for immersing the host animal.
 21. The delivery system of claim 20 wherein the algal cell is dried and aimed into a powder prior to being suspended in water.
 22. The delivery system of claim 7 wherein the algal cell is mixed with a liquid and packaged as a drink.
 23. The delivery system of claim 7 wherein the expression vector is incorporated into genetic material selected from the group consisting of nuclear, chloroplast, and mitochondrial.
 24. A method for inducing an immune response in an animal, comprising the steps of obtaining a transgenic alga expressing an antigenic peptide, and administering the transgenic alga to the animal.
 25. The method of claim 24 wherein the transgenic alga is administered by feeding.
 26. The method of claim 25 wherein the animal is a fish, and the immune response is detectable in a sample of serum from the fish.
 27. The method of claim 24 wherein the transgenic alga is administered by immersing the animal in a suspension comprising the alga.
 28. The method of claim 27 wherein the animal is a fish, and the immune response is detectable in a sample of mucus from the fish.
 29. A method for inducing enhanced growth in an animal, comprising the steps of obtaining a transgenic alga expressing a peptide derived from a growth hormone, and administering the transgenic alga to the animal.
 30. A method for controlling a pathogenic population in an animal, comprising the steps of obtaining a transgenic alga expressing a peptide derived from a bactericidal or insecticidal protein, and administering the transgenic alga to the animal. 